Orchestration of the cell division cycle includes a series of checkpoints which ensure that some events are completed before others begin (Murray, A. et al. The Cell Cycle Oxford University Press (1993). One set of controls determines whether cells replicate their genome in preparation for division (G1/S), while another checks that DNA replication is complete and that the cell has grown sufficiently for division to take place (G2/M) (Nasmyth, K. Science 274: 1643-1645 (1996). Cyclins and cyclin-dependent kinases (CDKs) regulate these events in part by controlling the transcription of specific effector genes (Okayama, H. et al., Adv. Cancer Res. 69: 17-62 (1996); Sanchez, I et al., Curr. Opin. Cell. Biol. 8: 318-824 (1996)). In budding yeast, CDC28 regulates the transcription of genes whose products are needed for the G1/S transition or S phase (Andrews, B. J. et al., J. Biol. Chem. 265: 14057-14060 (1990); Johnston, L. H. et al., Nucl. Acids Res. 20: 2403-2010 (1992)) via the transcription factors SBF (Swi4-Swi6) and DSC1 (Swi6-Mbp1) (Andrews, B. J. et al., Cell 57: 21-29 (1989); Dirick, L. et al., Nature 357: 508-513 (1992); Lowndes, N. F. et al., Nature 357: 505-508 (1992); Lowndes, N. F. et al., Nature 350: 247-250 (1991); Taba, M. R. et al., Genes Dev. 5: 2000-2013 (1991)). In fission yeast, the SBF-related heterodimer, MBF, is required for the expression of similar genes (Aves, S. J. et al., EMBO J. 4: 457-463 (1985); Lowndes, N. F. et al., Nature 357: 505-508 (1992); Tanaka, K. et al., EMBO J. 11: 4923-4932 (1992)). In mammalian cells, the CDK-regulated transcription factor E2F plays a key role in regulating the G1/S transition (Muller, R., Trends Genet. 11: 173-178 (1995); Sanchez, I et al., Curr Opin Cell. Biol. 8: 318-824 (1996)). E2F is complexed with Rb until Rb phosphorylation by G1 cyclin-dependent kinases releases E2F to activate transcription of immediate early genes including myc, fos, and jun (Beijersbergen, R. L. et al., Biochim. Biophys. Acta 1287: 103-120 (1996)).
Similar cyclin-dependent control mechanisms regulate the G2/M transition (Forsberg, S. L. et al., Annu. Rev. Cell. Biol. 7: 227-256 (1991); Nurse, P., Cell 79: 547-550 (1994); Nurse, P., Nature 344: 503-508 (1990)), but less is known about their downstream targets (Stukenberg, P. T. et al., Curr. Biol. 7: 338-348 (1997)). In fission yeast, regulation of the Cdc2-Cdc13 cyclin-dependent kinase-cyclin complex by the Weel kinase and Cdc25 phosphatase is thought to be the primary mechanism controlling G2/M (Okayama, H. et al., Adv. Cancer Res. 69: 17-62 (1996); Russell, P. et al., Cell 49: 559-567 (1987)). The Cdc2-Cdc13 complex accumulates during S phase, but Cdc2 is phosphorylated and thereby maintained in an inactive state by Weel (Fleig, U. N. et al., Semin. Cell. Biol. 2: 195-205 (1991); Lundgren, K. et al., Cell 65: 1111-1122 (1991)). As cells complete DNA replication, Weel is phosphorylated by Nim1 and thereby inactivated (Russell, P. et al., Cell 49: 559-567 (1987)), and Cdc25 accumulation leads to the dephosphorylation of Cdc2 (Gautier, J. et al., Cell 67: 197-211 (1991); Moreno, S. et al., Nature 344: 549-552 (1990)). Dephosphorylation and activation of Cdc2 heralds progression through G2 and entry into mitosis. The biochemical events controlling G2/M transit in mammalian cells are remarkably similar to those in S. pombe. Mammalian Cdc2 kinase accumulates in S phase (Shimizu, M. et al., Mol. Cell. Biol. 15: 2882-2892 (1995)) and is regulated by a Weel kinase (Parker, L. L. et al., Science 257: 1955-1957 (1992)) and Cdc25 phosphatase (Honda, R. et al., FEBS Lett. 318: 331-334 (1993)). While G2/M progression requires the coordinated expression of many genes, how this is regulated at the level of transcription remains largely unknown. The identification and characterization of transcription factors regulating G2 progression and mitotic entry, therefore, would significantly advance our understanding of the mechanisms controlling this portion of the cell cycle.
Most mammalian cells, such as hepatocytes, reside in G0 and can re-enter the cell cycle and undergo mitosis. Significant exceptions to this general rule include skeletal and cardiac myocytes, which are terminally differentiated and apparently incapable of undergoing mitosis shortly after the postnatal period. Tam et al. disclosed the possibility that reversal of terminal differentiation in cardiac myocytes might be achieved by manipulation of pocket proteins and/or cyclin D and cdk2 expression and function (Annals NY Acad Sci. 752: 72-79 (1995). Kirshenbaum et al. (J. Biol. Chem. 270: 7791-7794 (1995)) disclosed the reactivation of DNA synthesis, but not proliferation, of cardiac myocytes by the adenoviral protein E1A in concert with E1B delivered via an adenovirus vector. Kirshenbaum et al. (Dev. Biol. 179: 402-411 (1996)) disclosed that E2F-1 delivered via an adenovirus vector together with E1B can also activate DNA synthesis and cause the accumulation of cardiac myocytes in G2/M.
Thus, a need exists for agents which can direct cell cycle progression through G2 and entry into mitosis. The instant invention addresses this need and more.